UDPglucose pyrophosphorylase from Xanthomonas spp. Characterization of the enzyme kinetics, structure and inactivation related to oligomeric dissociation.
The genes encoding for UDPglucose pyrophosphorylase in two Xanthomonas spp. were cloned and overexpressed in Escherichia coli. After purification to electrophoretic homogeneity, the recombinant proteins were characterized, and both exhibited similar structural and kinetic properties. They were identified as dimeric proteins of molecular mass 60kDa, exhibiting relatively high specific activity ( approximately 80Units/mg) for UDPglucose synthesis. Both enzymes utilized UTP or TTP as substrate with similar affinity. The purified Xanthomonas enzyme was inactivated after dilution into the assay medium. Studies of crosslinking with the bifunctional lysyl reagent bisuberate suggest that inactivation occurs by enzyme dissociation to monomers. UTP effectively protects the enzyme against inactivation, from which a dissociation constant of 15microM was calculated for the interaction substrate-enzyme. The UTP binding to the enzyme would induce conformational changes in the protein, favoring the subunits interaction to form an active dimer. This view was reinforced by protein modeling of the Xanthomonas enzyme on the basis of the prokaryotic UDPglucose pyrophosphorylase crystallographic structure. The in silico approach pointed out two main critical regions in the enzyme involved in subunit-subunit interaction: the region surrounding the catalytic-substrate binding site and the C-term.
Bosco
M B
MB
Universidad Nacional del Litoral, Paraje "El Pozo", Santa Fe, Argentina.
Machtey
M
M
Iglesias
A A
AA
Aleanzi
M
M
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D017124:Conserved Sequence; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics; D005784:Gene Amplification; D005798:Genes, Bacterial; D005822:Genetic Vectors; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010957:Plasmids; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D014169:Transformation, Bacterial; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014974:Xanthomonas / Q000201:enzymology* / Q000235:genetics
D011994:Recombinant Proteins; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase